Mechanisms of Pin1 Regulation of IRAKM Stability in Toll-Like Receptor/Interleukin-1 Receptor Signaling

Abstract

The molecular mechanisms of asthma, a chronic inflammatory sickness that involves activation of the innate immunity signaling pathways, are not yet fully understood. Our studies focus on the peptidyl-prolyl isomerase enzyme Pin1 and its substrates in innate immunity signaling. Prolyl peptide bonds, such as in pS/T-P motifs, can exist in two distinct isomer conformations, cis and trans, that exchange on a slow time scale (several minutes). Isomerization of pS/T-P motifs can be accelerated by Pin1. Pin1 has recently been shown to be activated by IL-33 which activates IL-1R signaling in Th2 cells, inducing asthma. Pin1 knockout (KO -/-) abolishes Th2 cytokine release thereby preventing asthma symptoms. Pin1 also prevents degradation of IRAKM, a negative regulator of TLR/IL-1R signaling, but promotes the nuclear localization of IRAKM. IRAKM is phosphorylated upon IL-33 treatment. The phosphomimetic mutation IRAKM S110E is dramatically stabilized in a Pin1-dependent manner, even more than wild type IRAKM. We test the model that Pin1 regulation of IRAKM stability and nuclear localization is mediated by the direct interaction of Pin1 with phosphorylated IRAKM. The dissociation constants, KD, for Pin1 binding to phosphorylated IRAK-M (pIRAKM) and to the phosphomimetic mutation IRAKM S110E were determined using the 1H −15N HSQC NMR experiment. Furthermore, the Pin1 catalyzed isomerization of wild type pIRAKM and IRAKM S110E were measured using the ROESY NMR experiment. This study is expected to contribute to our understanding of the novel mechanism of Pin1 regulation of the TLR/IL-1R signaling pathway in terms of specific domains of IRAKM.